ABSTRACT Hematopoietic differentiation involves progression from the progenitor to precursor stages, and final maturation. Master Transcription factors (MTFs) such as GATA1 and GATA2 activate a critical cell-specific program, but additional transcription factors that drive stage-specific expression remain to be defined. Extracellular signals are transmitted to the nucleus, which activate signaling transcription factors (STFs). We studied human CD34 cells differentiated to the erythroid lineage, and examined the activation and binding of specific STFs to DNA representing several signaling pathways. We identified regions of the genome corresponding to stage-specific genes that are co-occupied by MTFs and STFs. We called these co-occupied regions ?transcriptional signaling centers? (TSCs) because they render the adjacent genes inducible by growth factors or small molecules. The BMP-signaling transcription factor SMAD1 is a marker of active TSCs and binds adjacent to GATA-factors to mark active genes at each stage of differentiation. SMAD1 is predictive of where other STFs bind, such as the cAMP-directed CREB, WNT-directed TCF7L2, and TGF?-directed SMAD2. Each ligand can activate (or repress) TSCs, leading to altered enhancer activity and gene expression. Co-binding of SMAD1 and GATA factors allows BMP induction of target genes, and mutation of a SMAD1-site in one TSC demonstrated a requirement of SMAD1-binding for appropriate gene expression. An examination of single nucleotide polymorphisms (SNPs) associated with erythroid traits demonstrates enrichment of such variations at TSCs, where many mutations occur at SMAD or other STF binding sites within the local region. The majority of human erythroid GWAS genes have mutations in STF binding sites in TSCs, but only a minority of SNPs affect the binding of MTFs. We showed that a polymorphism in a SMAD binding site within a TSC reduces SMAD1 binding based on gel mobility shift analysis and causes a specific reduction of expression of the associated gene in human blood cells. Other signals such as PGE2 also lead to activation of TSCs. We have shown that PGE2 induces stem cell birth during embryogenesis, and enhances hematopoietic stem cell (HSC) transplantability in fish, mice and humans. PGE2 enhanced HSCs are currently in a fourth clinical trial for patients with leukemia. Since the PGE2-stimulated STF CREB binds adjacent to SMAD1 in TSCs, we plan to examine if targets of both pathways are similar, or if specific gene programs are activated according to ligands. We will evaluate how PGE2 and BMP alter chromatin to lead to specific gene expression changes. Our data using micrococcal nuclease sensitivity studies suggest that within a few hours, there is a reorganization of chromatin resulting in greater accessibility of regions bound by the STFs. We plan to utilize ChIP-seq, ATAC- seq and protein biochemistry to examine how these chromatin alterations lead to gene expression changes. Understanding the specificity of signaling pathways and their impact on gene expression may lead to novel therapies for erythroid disorders including thalassemia and sickle cell anemia.! !